Low back pain (LBP) is the leading cause of disability worldwide, of significant socio-economic importance and is strongly associated with structural breakdown and degeneration of the intervertebral disc (IVD). Mechanical loading plays an essential role in maintaining the mechanical integrity of the IVD and multiple other spinal tissues through regulating biosynthesis of extracellular matrix (ECM) proteins. However, abnormal loading can alter this mechanical homeostasis and lead to further structural breakdown and degeneration. In this regard, understanding the magnitude of strains and pressures the cells experience within the tissue and the mechanisms through which cells sense these signals can provide insights into the regulation of IVD development and maintenance and inform biologic strategies for potential regeneration. This project utilizes novel DNA origami biosensors and microscale mass spectroscopy techniques to determine the role the specialized tissue immediately surrounding the cell, called the pericellular matrix (PCM), plays in transducing the physiochemical signals that embedded cells experience within the IVD. In particular, the osmotic environment engendered by the composition of the negatively charged and hydrated ECM within the IVD is an important physiochemical signal linking tissue loading and cellular metabolism. The PCM differs from the bulk ECM in its composition with greater concentrations of Perlecan and collagen type VI, which models have suggested induce greater osmotic pressures than in the bulk ECM. In order to understand how mechanical loads maintain homeostasis or promote pathophysiology there is a critical need to understand how osmotic pressures vary spatially in IVD tissue and to quantify the pressure cells experience in situ. We first propose to determine the minimum detectable ion gradient that novel DNA origami sensors can detect (Aim 1A) and determine if the sensor function is influenced by hydrostatic pressure (Aim 1B), which can also develop within loaded tissue. We then propose to utilize laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) to measure the ion concentrations within the PCM and ECM in healthy and degenerated IVD tissue and in cells isolated with intact PCM (Aim 2A). Finally, we will utilize DNA origami biosensors to examine the dynamic changes in pericellular ion concentrations under confined compression loading (Aim 2B). Together, these aims define and evaluate an entirely novel sensor technology to address the role of the PCM in regulating mechanobiology of the intervertebral disc. A detailed understanding of the magnitude of physiochemical signals that cells experience within loaded tissue is required into to develop a mechanistic understanding of mechanobiology and will provide critical insight into the development of regenerative strategies for LBP.